● ensure further and

HISTOPATHOLOGY complete hardening and

preservation of tissues.
Tissue processing ● Fixation
● Decalcification
● Dehydration Hollow organs should be packed with cotton
● Clearing soaked in fixative or completely
● Impregnation opened before being immersed in
● Embedding adequate fixing solution
● Trimming
● Sectioning Air-filled lungs organ may be covered with several
● Staining layers of gauze to maintain it under
● Mounting the surface
● Labeling
● Filling Frozen sections May lead to formation of ice crystal
artifacts
Purpose of 40% Fixative used to unprocessed
formalin specimen (prior to the start of tissue Hard tissues cervix, uterine, fibroids, skin,
processing; uncut specimens) fingernails, etc.

10% formalin ● It is prepared by mixing 40 washed out in running water

% Formaldehyde gas in 100 overnight and immersed in 4%
w/v of distilled water aqueous phenol solution for 1 – 3
● Used in fixation days. This will soften the tissue and
allow easier sectioning without
producing any marked distortion of
Fixation the preservation of solid tissue from the cell structures
destruction and post mortem
changes. DECALCIFICATION process of removing calcium and
lime salts and its deposits from hard
process by which constituents of tissues so that softening will take
cells and tissues are fixed in a place to facilitate embedding and
chemical so that they will withstand cutting.
treatment with various reagents
with minimum loss or DEHYDRATION Commonly used: ethyl alcohol
decomposition.
the process of removing
Tissue fixative ratio 1:20 intracellular and extracellular water
from the tissue following fixation
Factors affecting ● Size and thickness and prior to wax impregnation.
fixation ● Presence of mucus, fats
and blood CLEARING Commonly used: xylene/xylol
● Temperature (DEALCOHOLIZATIO
● Concentration N) process of removing excess alcohol
● Tissue fixative ratio from the tissue and replacing with a
substance that will dissolve the wax
which the tissue is to be
Methods removing ● Kardasewitch’s method impregnated.
pigments by ● Lillie’s method
formalin ● Picric acid clearing time 30 mins to 1 hour

IMPREGNATION Commonly used: paraffin wax

Secondary fixation ● improve the demonstration (INFILTRATION)
of particular substances. the process of completely removing
● make a special staining the clearing agent from the tissue
technique possible and replaced by a medium that will
secondary fixative acting as completely fill all the tissue cavities
mordant. and interstitial tissues
 Temperature: 2 degrees higher than ● Descending concentrations
its melting point of alcohol/ethyl alcohol
(Hydration of tissues;
EMBEDDING process by which the impregnated introduction of water to
(CASTING OR tissue is placed into a precisely tissues)
BLOCKING) arranged position in a mold ● Wash in running water (2
containing a medium which is then mins)
allowed to solidify ● Hematoxylin stain (basic
stain- stains nuclear; 10
!same medium as impregnation! dips then leave for 5
minutes)
Molds: kanang sudlanan ● Wash
● Eosin Y (30 dips; acidic
Trimming the excess wax is cut off from the stain- stains cytoplasm)
block to expose the tissue surface in ● Wash
preparation for actual cutting ● Ascending concentration of
alcohol- dehydration
Rotary Microtome Use: for cutting paraffin embedded ● Xylene
sections

Knife: plane concave Acid alcohol Cleaning medium for slides prior to
mounting
Ribbon size: 4-6 um
MOUNTING placing of cover glass on the
Freezing microtome Use: for cutting celloidin embedded medium
(Cryostat) sections
Cryostat / frozen The frozen section is the rapid tissue
Knife: biconcave section section by cooling the tissue with
the help of cryostat to provide
Ribbon size: 7-9 u immediate report of the tissue
sample. The cryostat is the
SECTIONING/ the process of cutting tissue into instrument to freeze the tissue and
CUTTING OF SECTIO uniformly thin slices measured in also to cut the frozen tissue for
micra (u) or millimicron (um) with microscopic sections. The rapid
the aid of a machine, to facilitate freezing of the tissue sample
the studies under the microscope converts the water into ice.

Thickness: 3-5um TURN AROUND TIME: 30 MINUTES

MINOR PROCESSES ● Floating – out (temp: 40-60 STAINING: same as fnab,cell cyto
AFTER SECTIONING: degrees or 6-10 degrees and paps (GCGMH)
below the melting point of
the wax) Cell block Processing of sediments, blood
● Application of Adhesive clots, or grossly visible tissue
● Fishing out fragments from cytological
● Orientation specimens into paraffin blocks that
● Deparaffinization- removal can be cut and stained by the same
of excess wax by the use of methods used in histopath
an alcohol lamp, or placed
in a hot oven at 60 degrees FNAB Technique which a fine needle is
centigrade for 15 to 30 introduced into a mass, cellular
mins., or dipping several material is aspirated, and a
times in xylene. cytological diagnosis is rendered

STAINING ● Put inside the oven for 10 Undergo cytological exam

minutes prior to staining
(melts wax) Core needle Obtains a cylinder of tissue about
● Xylene (final clearing) 1/16th of a diameter
 ● 5 dips in 2nd propano
Allow patho to perform a ● 5 dips xylene
histological study of tissue structure ● 5 dips in 2nd xylene
and cells ● Clean, mount and label

PAP smear Procedure to test for cervical cancer Polychromatic stain Consists of eosin y, bismarck brown
in women and, light green

Color acid groups blue (DNA/RNA),

PAP smear ● Fix in ether-ROH and pass basic groups orange (protein
procedure (MTAP thru 80% ROH, 40% ROH eosinophil) and metachromatic
notes) and distilled H2O. substances violet (mast cell and
● Stain in Harris Hematoxylin basophilic granules)
for 4-5 minutes.
● Wash with H2O. Valuable stain for blood cell diff
● Pass thru 0.25% HCl in 50% count and evaluation
ROH.
● Immerse in 1.5% NH4OH in CELL BLOCK: ● Centrifuge specimen for 5
70% ROH for 1 minute. PARAFFIN METHOD mins
● Rinse in 70% ROH and pass ● Discard supernatant
thru 80% and 95% ROH. ● Add 95% ethanol to
● Stain with OG 6 for 1.5-a sediments (3ml)
minutes. ● Mix and stand for few mins
● Pass thru 3 changes of 95% ● Centri for 5 mins
ROH. ● Place sediments in a filter
● Stain with EA 65 or EA 50 paper
for 3 minutes. ● Process samples as in
● Pass thru 3 changes of 95% routine tissue processing
ROH.
● Dehydrate and clear in:
○ absolute ROH, CELL BLOCK: SMEAR ● Shake specimen bottle to
○ equal parts of METHOD resuspend sediments
ether and absolute ● Take an aliquot, about 10
ROH, ml into test tube
○ 2 changes of xylol ● Centrifuge, decant
● Mount in Canada Balsam. supernatant, make 2-3
smears (pull apart method)
Results: ● Fix smear with 95% ethanol
● Cytoplasm – either bright
red or greenish blue
● vesicular nucleus – blue
● pyknotic nucleus – dark
blue to black
● bacteria – dark blue
● mycelia – violet
● Trichimonas vaginalis –
pale greenish blue blob of
cytoplasm

FNAB, cell cyto, ● Fix (95% alcohol)

PAPs stain ● Hematoxylin 5-10 dips and
procedure leave for 1 min
(GCGMH) ● Wash with running water
● Propanol 3-5 dips
● Polychromatic stain 5-10
dips and leave for 1 min
● Wash
● 5 dips propanol